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Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM - J. Biol. Chem. (2009)

Bottom Line: At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer.RTA is then stably bound to the membrane because it is nonextractable with carbonate.Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843-1114, USA.

ABSTRACT
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

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Related in: MedlinePlus

Secondary structural changes in RTA and saporin. Recordings of the far-UV CD spectra of RTA and saporin, each 5 μm in buffer C at 37 °C, in the absence or presence of 200 μm liposomes containing different percentages of PC or PS. The scans were corrected by the subtraction of blanks containing only buffer and/or liposomes. The panels show the averaged spectra of at least two experiments.
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fig7: Secondary structural changes in RTA and saporin. Recordings of the far-UV CD spectra of RTA and saporin, each 5 μm in buffer C at 37 °C, in the absence or presence of 200 μm liposomes containing different percentages of PC or PS. The scans were corrected by the subtraction of blanks containing only buffer and/or liposomes. The panels show the averaged spectra of at least two experiments.

Mentions: The Secondary Structure of RTA Is Altered upon Binding to Lipids—To determine whether the binding of RTA to membranes introduces a change in its secondary structure, we compared the CD spectra of unmodified RTA in the absence and presence of liposomes at 37 °C. The CD spectrum of RTA indicates a significant amount of α-helix with minima at 208 and 222 nm (Fig. 7A), consistent with the published crystal structure (43). Upon exposure to PCPS liposomes, the conformation of RTA changes and its α-helical structure is reduced (Fig. 7C) concomitant with an increase in β sheet. The CD data therefore show that some α-helical structure is lost upon RTA binding to membranes. In contrast, the secondary structure of saporin was not altered by PCPS liposomes (Fig. 7, compare D and E). The RTA secondary structure was unchanged by incubation at 30 °C in the presence of PCPS liposomes (Fig. 7C), although increasing the proportion of PS in the liposomes promoted the loss of α-helical structure of RTA at lower temperatures (supplemental Fig. S4). Thus, the secondary structural change detected by CD requires both binding to a PS-containing membrane and a temperature of 37 °C.


Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C.

Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM - J. Biol. Chem. (2009)

Secondary structural changes in RTA and saporin. Recordings of the far-UV CD spectra of RTA and saporin, each 5 μm in buffer C at 37 °C, in the absence or presence of 200 μm liposomes containing different percentages of PC or PS. The scans were corrected by the subtraction of blanks containing only buffer and/or liposomes. The panels show the averaged spectra of at least two experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665077&req=5

fig7: Secondary structural changes in RTA and saporin. Recordings of the far-UV CD spectra of RTA and saporin, each 5 μm in buffer C at 37 °C, in the absence or presence of 200 μm liposomes containing different percentages of PC or PS. The scans were corrected by the subtraction of blanks containing only buffer and/or liposomes. The panels show the averaged spectra of at least two experiments.
Mentions: The Secondary Structure of RTA Is Altered upon Binding to Lipids—To determine whether the binding of RTA to membranes introduces a change in its secondary structure, we compared the CD spectra of unmodified RTA in the absence and presence of liposomes at 37 °C. The CD spectrum of RTA indicates a significant amount of α-helix with minima at 208 and 222 nm (Fig. 7A), consistent with the published crystal structure (43). Upon exposure to PCPS liposomes, the conformation of RTA changes and its α-helical structure is reduced (Fig. 7C) concomitant with an increase in β sheet. The CD data therefore show that some α-helical structure is lost upon RTA binding to membranes. In contrast, the secondary structure of saporin was not altered by PCPS liposomes (Fig. 7, compare D and E). The RTA secondary structure was unchanged by incubation at 30 °C in the presence of PCPS liposomes (Fig. 7C), although increasing the proportion of PS in the liposomes promoted the loss of α-helical structure of RTA at lower temperatures (supplemental Fig. S4). Thus, the secondary structural change detected by CD requires both binding to a PS-containing membrane and a temperature of 37 °C.

Bottom Line: At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer.RTA is then stably bound to the membrane because it is nonextractable with carbonate.Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas 77843-1114, USA.

ABSTRACT
After endocytic uptake by mammalian cells, the heterodimeric plant toxin ricin is transported to the endoplasmic reticulum (ER), where the ricin A chain (RTA) must cross the ER membrane to reach its ribosomal substrates. Here, using gel filtration chromatography, sedimentation, fluorescence, fluorescence resonance energy transfer, and circular dichroism, we show that both fluorescently labeled and unlabeled RTA bind both to ER microsomal membranes and to negatively charged liposomes. The binding of RTA to the membrane at 0-30 degrees C exposes certain RTA residues to the nonpolar lipid core of the bilayer with little change in the secondary structure of the protein. However, major structural rearrangements in RTA occur when the temperature is increased. At 37 degrees C, membrane-bound toxin loses some of its helical content, and its C terminus moves closer to the membrane surface where it inserts into the bilayer. RTA is then stably bound to the membrane because it is nonextractable with carbonate. The sharp temperature dependence of the structural changes does not coincide with a lipid phase change because little change in fluorescence-detected membrane mobility occurred between 30 and 37 degrees C. Instead, the structural rearrangements may precede or initiate toxin retrotranslocation through the ER membrane to the cytosol. The sharp temperature dependence of these changes in RTA further suggests that they occur optimally in mammalian targets of the plant toxin.

Show MeSH
Related in: MedlinePlus